Nitric oxide (NO) mediates relaxation of vascular smooth muscle cells (SMCs), inhibits platelet and leukocyte adhesion, and prevents SMC proliferation. Delivery of NO to sites prone to intimal hyperplasia may prevent vascular occlusion following surgical bypass or angioplasty. We propose that one method to deliver NO locally is to transfer the inducible NO synthase (NOS-2) gene to sites of vascular injury. Unlike other NOS isoforms, NOS-2 produces large amounts of NO independent of calcium fluxes. These theoretical advantages should permit sustained NO synthesis. We have already established the feasibility of NOS-2 gene transfer using retroviral; and adenoviral; vectors in cultured SMCs, arterial segments, and in a rodent vascular injury model showing dramatic inhibition of intimal hyperplasia. These preliminary studies also reveal that functional NOS-2 expression is variably dependent on the cofactor tetrahydrobiopterin (BH4). We propose studies to fully test our hypothesis that NOS-2 gene transfer will prevent restenosis following vascular trauma associated with conventional treatments. In this proposal, we will pursue two interrelated aims to study the requirements for successful NOS-2 gene transfer. AIM I: To determine the efficacy and optimal conditions for in vivo NOS-2 gene transfer for the prevention of injury-induced intimal hyperlasia. We will begin by determining the optimal conditions for in vivo NOS-2 gene transfer needed to inhibit intimal hyperplasia by varying viral titer, time of viral exposure, and BH4 supplementation. NOS-2 gene transfer will also be tested in a pig vascular injury model, more relevant for human disease, in the face of atherosclerotic lesions. If BH4 supplementation is necessary, we will establish the efficacy of concurrently transferring GTP cyclohydrolase I (the rate limiting enzyme in BH4 biosynthesis) to supply BH4. We will examine the effect of NOS-2 expression and NO synthesis on vascular responses such as reedothelialization, vasomotor reactivity, and tensile strength. AIM I: To determine the molecular mechanisms by which nitric oxide inhibits intimal hyperlasia. In this AIM, we will elucidate the pathways by which NO prevents restenosis utilizing the optimal conditions for NOS-2 gene transfer established under AIM I. The effect of NOS-2 on platelets and leukocytes will be examined by the attachment of radiolabeled cells to injured arteries. We will determine if NOS-2 directly affects SMC proliferation and migration in vivo. Likely mechanisms for NO action in the inhibiting intimal hyperplasia will also be examined, ranging from the induction of apoptosis to the induction of inhibitory cell cycle genes (ie., p53 or p21) to extracellular matrix synthesis. At the completion of our studies, we will have characterized the mechanisms and conditions for effective NOS-2 gene transfer for the prevention of restenosis. This information will direct the development of this technology for the treatment of vasoocclusive disease and complications of conventional therapies.